A variety of cardiac disorders have an underlying genetic cause. For example, dilated cardiomyopathy (DCM) is a cardiac disease characterized by ventricular dilatation and systolic dysfunction. DCM is the most common cause of heart failure after coronary artery disease and hypertension, as well as the leading indication for heart transplantations. The cost for management of DCM in the US alone has been estimated at between $4 and $10 billion. Another important condition for therapy is hypertrophic cardiomyopathy (HCM), in which the sarcomeres replicate, causing cardiomyocytes to increase in size. In addition, the normal alignment of cardiomyocytes is disrupted, a phenomenon known as myocardial disarray. HCM is most commonly due to a mutation in one of nine sarcomeric genes.
Mutations in genes encoding sarcomeric, cytoskeletal, mitochondrial, and nuclear membrane proteins, as well as proteins involved in calcium metabolism, are associated with approximately a third to half the cases of DCM. Cardiac troponin T (cTnT) is one of the 3 subunits of the troponin complex (Troponin T, C, and I) that regulate the sarcomeric thin filament activity and muscle contraction in cardiomyocytes (CMs). cTnT is essential for sarcomere assembly, contraction, and force production. Mutations in the cardiac troponin T gene (TNNT2) often lead to DCM and are frequently expressed as a malignant phenotype with sudden cardiac death and heart failure at an early age. In vitro biochemical studies have found that decreased Ca2+ sensitivity and/or ATPase activity, which lead to impaired force production, may be the underlying mechanisms for certain TNNT2-mutation induced DCM.
Mouse models of TNNT2 mutations recapitulate the human DCM phenotype and have provided extensive insight into the possible mechanisms of the disease. However, several differences exist between the mouse and human models. For example, mouse resting heart rate is approximately 10-fold faster than human. The electrical properties, ion channel contributions, and cardiac development of mouse CMs are also different from those of human. The lack of complex intracellular interactions within cardiomyocytes for in vitro biochemical assays and species differences for mouse models undercut the value of these methodologies for understanding the cellular and physiological processes of DCM as well as for drug screening.
In addition, cardiac tissues from DCM patients are difficult to obtain and do not survive in long-term culture. Effective cellular models for dilated cardiomyopathy and other genetic cardiac conditions are of great interest for screening and development of effective therapies.
Pharmaceutical drug discovery, a multi-billion dollar industry, involves the identification and validation of therapeutic targets, as well as the identification and optimization of lead compounds. The explosion in numbers of potential new targets and chemical entities resulting from genomics and combinatorial chemistry approaches over the past few years has placed enormous pressure on screening programs. The rewards for identification of a useful drug are enormous, but the percentages of hits from any screening program are generally very low. Desirable compound screening methods solve this problem by both allowing for a high throughput so that many individual compounds can be tested; and by providing biologically relevant information so that there is a good correlation between the information generated by the screening assay and the pharmaceutical effectiveness of the compound.
Some of the more important features for pharmaceutical effectiveness are specificity for the targeted cell or disease, a lack of toxicity at relevant dosages, and specific activity of the compound against its molecular target. The present invention addresses this issue.
Publications.
Methods to reprogram primate differentiated somatic cells to a pluripotent state include differentiated somatic cell nuclear transfer, differentiated somatic cell fusion with pluripotent stem cells and direct reprogramming to produce induced pluripotent stem cells (iPS cells) (Takahashi K, et al. (2007) Cell 131:861-872; Park I H, et al. (2008) Nature 451:141-146; Yu J, et al. (2007) Science 318:1917-1920; Kim D, et al. (2009) Cell Stem Cell 4:472-476; Soldner F, et al. (2009) Cell. 136:964-977; Huangfu D, et al. (2008) Nature Biotechnology 26:1269-1275; Li W, et al. (2009) Cell Stem Cell 4:16-19).
Additional publications of interest include Stadtfeld et al. Science 322, 945-949 (2008); Okita et al. Science 322, 949-953 (2008); Kaji et al. Nature 458, 771-775 (2009); Soldner et al. Cell 136, 964-977 (2009); Woltjen et al. Nature 458, 766-770 (2009); Yu et al. Science (2009).